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Restriction enzyme SmaI and expression purification method thereof

A restriction enzyme, expression and purification technology, applied in the field of restriction enzyme SmaI and its expression and purification, can solve the problems of low protein yield, cumbersome separation and purification procedures, and high price

Pending Publication Date: 2019-10-29
莫纳(武汉)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to problems such as low expression, cumbersome separation and purification procedures, and low protein yield, the currently commercially available SmaI restriction endonuclease is not only expensive, but also limits its wide application and in-depth research.
At the same time, a series of problems mentioned above also limit the in-depth research of many other widely used restriction endonucleases

Method used

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  • Restriction enzyme SmaI and expression purification method thereof
  • Restriction enzyme SmaI and expression purification method thereof
  • Restriction enzyme SmaI and expression purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] (1) Construction of recombinant engineering bacteria containing SmaI enzyme expression fragment

[0105] Using the nucleotide sequence shown in SEQ ID NO.1 as an amplification template, adding an upstream primer (SEQ ID NO.2) and a downstream primer (SEQ ID NO.3) to perform PCR amplification to obtain a PCR amplification product. The obtained PCR amplification product was double-digested with NcoI enzyme and XhoI enzyme to obtain the expression fragment of SmaI enzyme. Then the SmaI enzyme expression fragment is cloned into the pG-28b vector that has been cut with NcoI enzyme and XhoI enzyme to obtain the recombinant vector pG-28b-SmaI, such as Figure 8 shown. Transfer the recombinant vector pG-28b-SmaI into DH5α competent cells and extract the plasmid for sequencing. After the sequencing is correct, transform the above plasmid into ER2566 to obtain the recombinant engineering bacteria containing the SmaI enzyme expression fragment. Using appropriate aseptic techniqu...

Embodiment 2

[0118] The purification method of the SmaI enzyme in Example 2 is roughly the same as the expression and purification method in Example 1. The recombinant engineering bacteria containing the SmaI enzyme expression fragment constructed and preserved in Example 1 were used for activation and expression, the expressed bacteria were collected by centrifugation, and then the bacteria were lysed to collect the lysate.

[0119] The difference from Example 1 is that the order of ion exchange chromatography and heparin affinity chromatography is different, and the heparin affinity chromatography is passed first, followed by the ion exchange chromatography column. In this case, the dilution factor will be changed, increasing It increases the difficulty of diluting the sample, and increases the sample volume to a certain extent, and the purification time will be greatly increased.

[0120] SDS-PAGE analysis of samples from various stages in the process of purifying restriction enzyme Sma...

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Abstract

The invention provides an expression purification method restriction enzyme SmaI. The method comprises the following steps that recombinant bacteria containing restriction enzyme SmaI expression fragments are constructed, induced culture is conducted, bacterium bodies are fragmented, a product is purified and recycled, and the restriction enzyme SmaI is obtained; and the purifying modes comprise combination of at least two of Ni ion affinity chromatography, anion exchange chromatography or heparin-affinity chromatography. Through combination of multiple purifying modes, the restriction enzymeSmaI with the purity larger than 98% of obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a restriction endonuclease SmaI and an expression and purification method thereof. Background technique [0002] In organisms, there is a class of enzymes that can cut foreign DNA. It can limit the invasion of heterologous DNA and make it lose its vitality, but it has no damage to its own DNA, so that it can protect the original genetic information of cells. role. Because this cutting effect is carried out inside the DNA molecule, it is named restriction endonuclease (restriction enzyme for short). Its discovery led to the birth of DNA recombination technology, which greatly promoted the development of modern molecular biology and genetic engineering, and is an indispensable basic tool for contemporary genetic engineering research. [0003] With the discovery of the phenomenon of restriction modification, scientists discovered restriction endonucleases, and clarified the role of rest...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/22C12N15/70
Inventor 程槐旭胡梦竹燕东平聂尚海
Owner 莫纳(武汉)生物科技有限公司
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